Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells.

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence, this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes, Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution, the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay, which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2% vs. 9.9%, p > 0.05). However, when 100 mM Z-VAD-FMK was added to the post-thaw culture media, there was a significant enhancement in the survival rate from 9.9% to 14.4% (p < 0.05), which was further increased to 18.7% when Z-VAD-FMK was also added to the freezing solution as well (p < 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture.